Characterization of microvilli morphology and P-gp localization in confluent MDCKII-hMDR1-NKI and Caco-2 cell monolayers by three-dimensional structured illumination microscopy. Representative images of microvilli, as seen by filamentous actin staining (green) and P-gp staining (red) in Caco-2 cells (A, C) and MDCKII-hMDR1-NKI cells (B, D). (A and B) Left panels show the maximum intensity z-projections; rectangle panels next to the left panels show the orthogonal (yz) views obtained from the vertical yellow lines shown in the z-projections. Panels on the right are high magnification images obtained from the red rectangles. (C and D) All panels show the maximum intensity z-projection; the panels of second row show the high magnification images obtained from the purple square.

Cartoon figures of Caco-2 and MDCKII-hMDR1-NKI cells with microvilli and P-gp (dots). Red dots represent efflux active P-gp that can send their drug directly to apical chamber, whereas black dots represent efflux inactive P-gp. The morphology of microvilli was drawn approximately based on results from super-resolution microscopy, as shown in Fig. 2. In mouse small intestine, there is about 50 nm between adjacent microvilli, with some variability in the size of the aqueous space between them (Crawley et al., 2014). Microvilli of Caco-2 cells are more packed and regular than the ones in MDCKII-hMDR1-NKI cells. Differently from Caco-2 cells, microvilli on MDCKII-hMDR1-NKI cells are not all standing straight up; many of them are bent. Previously we proposed that the amphipathic P-gp substrate drug released at the base of a microvillus would be interacting with the same or a neighboring microvillus and the P-gp they contain (Bentz et al., 2005; Tran et al., 2005). Only the P-gp at the tips and at other apical chamber-exposed surfaces of the MDCKII-hMDR1-NKI bent microvilli can efflux the drug directly into the apical chamber with higher probability. According to this hypothesis, a larger fraction of total P-gp would be efflux active in MDCKII-hMDR1-NKI (red dots) when the total P-gp levels are similar among the two cell lines.

↵b Because peptide AGAVAEEVLAAIR yielded the higher amount of P-gp in both MDCKII-hMDR1-NKI and Caco-2 cells, we assume that this difference was due to different trypsin digestion efficiencies. The absolute amounts of P-gp used in this table are based on peptide AGAVAEEVLAAIR.